Phosphorescent Meso-Unsubstituted Metallo-Porphyrin Probe Molecules for Measuring Oxygen and Imaging Methods

ABSTRACT

Oxygen levels in biological tissue or systems can be measured by the phosphorescence quenching method using phosphorescent porphyrin probes, also referred to as a dendritic oxygen probes, with controllable quenching parameters and defined biodistributions. Provided are a “next generation” of oxygen sensors with substantially improved phosphorescence emission for better imaging capabilities, ease of use, increasing the quantum efficiency (phosphorescence intensity) and extending their range of applicability including constructing a class of oxygen sensors for making measurements in organic media. In addition, provided are methods for synthesizing new porphyrin constructs in which the porphyrin is made less flexible and more planar, changing with decrease internal quenching, and thereby increasing the phosphorescence emission used for oxygen sensing. Additional methods are provided for structurally modifying the dendrimer used to encapsulate the porphyrin phosphor to provide internal quenching of singlet oxygen molecules formed during oxygen measurements.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention is a continuation-in-part of, and claims priorityto, U.S. Non-Provisional patent application Ser. No. 13/778,777, filedon Feb. 27, 2013, which, in turn, claimed priority to U.S. ProvisionalApplication Ser. No. 61/603,668, filed on Feb. 27, 2012, both of whichare incorporated herein in their entirety.

GOVERNMENT INTEREST

This invention was supported in part by funds from the U.S. Government(National Institutes of Health) Grant No. HL0812273, R01EB007279,EB018464, and NS092986, and the U.S. Government may therefore havecertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to highly accurate and versatile opticalmethod for oxygen measurement, particularly useful for applications inliving human and animal tissue, and more particularly to novelphosphorescent probe molecules that possess brighter signal,well-defined chemical composition, and can potentially be used inclinics.

BACKGROUND OF THE INVENTION

The reliable and accurate measurement of oxygen supply in mammaliantissue is important to ensure that the oxygen supply is adequate. Thecirculatory system employs special oxygen-carrying protein molecules inred blood cells (hemoglobin) to deliver oxygen from the lungs throughoutthe body. Once dissociated from hemoglobin, oxygen is delivered to itsconsumption sites in cells by diffusion. It is the measurement of thisdissolved unbound oxygen that is most critical for quantifying tissuephysiological status. The phosphorescence quenching method underpinningthis patent application is unique in its ability to accomplish such ameasurement. This method is based on use of special phosphorescent probemolecules, which report on oxygen concentration in the environment withhigh specificity and accuracy.

Measurements of oxygen by phosphorescence quenching provide informationon oxygen consumption by the tissue, and through that, allow evaluationof tissue diseased states. Oxygen is a key metabolite, and tissuehypoxia is a critical parameter with respect to various tissuepathologies, such as retinal diseases (Berkowitz et al., Invest.Ophthalmol. Visual Sci. 40:2100-2105 (1999); Linsenmeier et al.,Ophthalmol. Visual Sci. 39:1647-1657 (1998)), brain abnormalities(Vannucci et al., J. Exp. Biol. 207:3149-3154 (2004); Brunel et al., J.Neuroradiology 31:123-137 (2004); Johnston et al., Neuroscientist8:212-220 (2002)), and cancer (Evans et al., J. Appl. Physiol.98:1503-1510 (2005)). Different oxygen levels in tissues can beindicative of tissue structure abnormalities, defects, whether causedexternally, or by genetic manifestations, resulting from disease.

Imaging tissue oxygen in vivo presents a challenging and importantproblem. Nevertheless, currently developing imaging technologies formapping tissue oxygenation (Rajendran et al., Radiol. Clin. North Am.43:169-187 (2005)) (e.g., NMR/EPR (Subramanian et al. NMR Biomed.17:263-294 (2004)), PET (Piert et al., Nucl. Med. 46:106-113 (2005);Apisarnthanarax et al., Rad. Res. 163:1-25 (2005)), near infraredtomographic techniques (Fenton et al., Brit. J. Cancer 79:464-471(1999); Liu et al., Appl. Opt. 39:5231-5243 (2000)), etc (Ballinger,Sem. Nucl. Med. 31:321-329 (2001); Foo et al., Mol. Imag. Biol.6:291-305 (2004)) suffer from many deficiencies, including invasiveness,low spatial and/or temporal resolution, lack of absolute calibration,poor specificity, etc., and remain yet to be adequately developed.

The phosphorescence quenching method (Vanderkooi et al., J. Biol. Chem.262:5476-5483 (1987); Wilson & Vinogradov, In: Handbook of BiomedicalFluorescence. Mycek M-A, Pogue B W, eds. Marcel Dekker; New York: 2003.Ch. 17) is superior in its ability to directly detect oxygen in tissue.A detailed summary is presented by Vinogradov & Wilson (2012)“Porphyrin-dendrimers as biological oxygen sensors,” In DesigningDendrimers (Capagna, Ceroni, Eds.), Wiley, New York) following theeffective filing date of this invention. When a phosphorescent probe isdissolved in the blood and excited using appropriate illumination, itsphosphorescence lifetime and intensity become robust indicators ofoxygen concentration in the environment. Phosphorescence quenching isexquisitely sensitive and selective to oxygen, possesses excellenttemporal resolution and can be implemented for high-resolution hypoxiaimaging in 2D (Rumsey et al., Science. 241:1649-1652 (1988); Vinogradovet al., Biophys. J. 70:1609-1617 (1996); Shonat et al., Annal. Biomed.Eng. 31:1084-1096 (2003)).

Efforts to develop 3D near infrared tomographic modality for mappingtissue oxygenation using phosphorescences, include Soloviev et al.,Applied Optics 42:113 (2003); Soloviev et al., Applied Optics 43:564(2004); Apreleva et al., Optics Letters 31:1082 (2006); Apreleva et al.,Applied Optics 45:8547 (2006); Apreleva et al., Optics Letters 33:782(2008), and recent clinically relevant developments of Cerenkovradiation-induced phosphorescence (Zhang et al., Biomedical OpticsExpress 3:2381 (2012). But highly accurate and versatile methods formeasuring oxygen remain to be further developed.

For phosphorescent compounds to be suitable for use as a phosphorescentoxygen probe (aka “phosphor” or “oxyphor”) in determination of tissueoxygenation, it is desirable that the compounds have (1) high absorbancein the near infrared region of the spectrum where natural chromophoresof tissue, such as hemoglobin or myoglobin, have only very weakabsorption; (2) phosphorescence with high quantum yields at roomtemperature, preferably greater than 0.05; and (3) suitable lifetimes,preferably from about 0.3 to about 1 msec.

A new class of phosphors suitable for oxygen measurement was previouslyreported in Vinogradov and Wilson, J. Chem. Soc., Perkin Trans. 2,103-111 (1995), and in U.S. Pat. No. 4,947,850, “Method and Apparatusfor Imaging an Internal Body Portion of a Host Animal,” by Vanderkooiand Wilson), and U.S. Pat. No. 5,837,865, “Phosphorescent DendriticMacromolecular Compounds for Imaging Tissue Oxygen,” by Vinogradov andWilson), which are incorporated herein by reference. In the general, thephosphorescent probes for oxygen measurements comprise three functionalparts: 1) phosphorescent core; 2) encapsulating and protecting ligandsand 3) the hydrophilic outer layer, which is usually made ofmonomethyloligoethyleneglycol or simply polyethyleneglycol (PEG)residues. Parts 2 and 3 comprise the so-called immediate “surroundingenvironment” of the phosphorescent core chromophore.

The functions of the three parts are as follows: a) phosphorescent coreprovides optical signal (phosphorescence), inducible by red/nearinfrared excitation sources and responsive to changes in the partialpressure of oxygen (pO₂); b) encapsulating ligands allow tuning of thecore accessibility to oxygen to optimize probe's sensitivity in thephysiological pO₂ range; and c) outer layer provides solubility andisolates the probe from interactions with endogenous biological species(proteins, nucleic acids, membranes etc) in order to maintain thecalibration constants for quantitative pO₂ measurements in biologicalenvironments.

Both aforementioned patents teach compounds based on complexes ofmetals, such as Pd and Pt, with porphyrins and aromatically π-extendedporphyrins, such as, for example, tetrabenzoporphyrin,tetranaphthaloporphyrin, tetraanthraporphrin and various derivativesthereof, which play the role of phosphorescent cores (part 1). Thesecomplexes possess bright room temperature phosphorescence, and Pd and Ptcomplexes of tetrabenzoporphyrins and tetranaphthaloporphyrins areespecially desirable because they show strong light absorption in thenear IR region (610-650 nm and 700-720 nm, respectively), where tissueis practically transparent. Moreover, Pd tetrabenzoporphyrins (PdTBP)and their derivatives have been shown to have long-lived phosphorescence(˜250 μsec) with quantum yields of 0.08-0.10%. These values have beenlater re-measured against improved fluorescence standards usedthroughout the remainder of this specification, and shown to be0.0015-0.04 (see Esipova et al, Anal. Chem. 83:8756 (published on-lineOct. 11, 2011).

Generally, the surrounding environment determines properties of thephosphorescent probe with respect to oxygen measurement, including watersolubility, toxicity, oxygen quenching constant, sensitivity of themeasurements to chemically active components of tissue, and ease ofexcretion of the probe from the body through the kidney. It is alsodesirable to design the surrounding environment, such that it comprisesan inert globular structure around the phosphor, through which onlysmall uncharged molecules, i.e., oxygen, can diffuse into the closevicinity of the phosphorescent core for efficient quenching.

The '865 patent above teaches that the optimal surrounding environmentfor the phosphorescent core is made of dendrons as encapsulating ligands(part 2) and polyethyleneglycols (or oligoethyleneglycols) as the outerlayer of the probe (part 3). (Note that together the encapsulatingdendrons are said to comprise a dendrimer. Accordingly, thecorresponding phosphorescent probes are termed dendritic.) Dendriticprobes so far have shown to be superior phosphors for oxygenmeasurements in biological systems. Many laboratories around the worldcurrently use these molecules for oxygen measurements in the blood,tissue interstitial space, various organs and with application ofdifferent modes of the phosphorescence quenching method (see above).See, e.g., Sakadzic et al., Nat. Methods 7:755 (2010); Devor et al., J.Neuroscience 31:13676 (2011); Lecoq et al., Nature Medicine 17:893(2011); and since the effective filing date of this application,Parpaleix et al., Nature Medicine 19:241-246 (2013), which havecapitalized on the use of the dendritic oxygen probes to decipher brainenergy metabolism in the field of neuroscience. Thus the prior andpresent art have clearly established tremendous value of dendriticoxygen probes, warranting their further improvement and optimization.

It has therefore been an ongoing need in the art to further improve onthe structure of dendritic phosphorescent probes by altering andimproving their chemical structure, thereby providing a “nextgeneration” of oxygen sensors with substantially improvedphosphorescence emission for better imaging capabilities, ease of use,and range of applicability. In addition, some of the new molecules inthis disclosure provide measurements of oxygen, not only in an aqueousenvironment, but also in liquid organic media, such as organic solventsand/or oils.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide phosphors comprisinga new class of porphyrin-based oxyphors and schemes for synthesizing thesame, wherein the porphyrin is made less flexible and more planar, twomodifications that the inventors have shown significantly (5-10 fold)increases the quantum yield over the prior art probes, and thereforeserve as the bases for significantly improved oxygen sensors. Thesestructurally improved, extended porphyrins are then complexed withdendrimers to surround the phosphorescent core by supramolecularstructures which are designed to be highly soluble in either water basedmedia or in liquid organic media, such as organic solvents and/or oils.They provide additional sought-after characteristics of phosphorescentprobes such as long-lived phosphorescence and high quantum yields.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description, examples and figures whichfollow, all of which are intended to be for illustrative purposes only,and not intended in any way to limit the invention, and in part willbecome apparent to those skilled in the art on examination of thefollowing, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. It should be understood, however, that theinvention is not limited to the precise arrangements andinstrumentalities shown.

FIG. 1 illustrates an exemplary synthesis of meso-unsubstituted TBP,wherein M is an element selected from H₂, Zn, Ni, CU, Pd, Pt, Ru, Au,Os, Ir, La. Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu.

FIG. 2 depicts DAPIP and TAPIP porphyrins resulting from the disclosedsynthesis scheme, with element M being selected from H₂, Zn, Ni, CU, Pd,Pt, Ru, Au, Os, Ir, La. Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm,Yb, and Lu.

FIG. 3 depicts the synthesis of meso-unsubstituted TAPIP.

FIG. 4 depicts the synthesis of meso-unsubstituted DAPIP.

FIGS. 5A-H depict the absorption and emission spectra of selected metalcomplexes of TAPIP and DAPIP in dimethylformamide at 22 degrees Celsius;FIG. 5A shows the absorption spectra of ZnTAPIP; FIG. 5B shows thefluorescence of ZnTAPIP; FIG. 5C shows the absorption spectra ofPdTAPIP; FIG. 5D shows the phosphorescence of PdTAPIP; FIG. 5E shows theabsorption spectra of PtTAPIP; FIG. 5F shows the phosphorescence ofPtTAPIP; FIG. 5G shows the absorption of PtDAPIP: FIG. 5H shows thephosphorescence of PtDAPIP.

FIG. 6 depicts the 2PA spectra of PtTAPIP as a solid line and the 2PAspectra of PdTAPIP as a dashed line, both in dimethylacetamide at 22degrees Celsius.

FIG. 7 illustrates exemplary dendrimerization of porphyrins: i) peptidecoupling; ii) hydrolysis.

FIG. 8 illustrates an exemplary dendritic phosphorescent probe modifiedwith hydrophobic residues to allow oxygen measurements in liquid organicsolutions, e.g., saturated alkanes, mineral oils etc., wherein M is anelement selected from H₂, Zn, Ni, CU, Pd, Pt, Ru, Au, Os, Ir, La. Ce,Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

The present invention provides highly efficient and highly solublephosphorescent probes suitable for measurements of oxygen in tissue ofanimals and humans. Inventive probes are surrounded by an inert globularstructure, an example of which is derivatized PdTBP (Pdtetrabenzoporphyrins) surrounded by three-dimensional supramolecularstructure known as a “dendrimer,” which is well understood in the field.

Oxygen levels in biological systems can thus be measured by thephosphorescence quenching method using probes with controllablequenching parameters and defined biodistributions. A general approach isprovided to the construction of phosphorescent nanosensors with tunablespectral characteristics, variable degrees of quenching, and a highselectivity for oxygen that are soluble in aqueous or organic solvents(e.g., benzene, toluene, hexane, octane, tetrahydrofurane, mineral oil,and the like), permitting phosphorescence measurements throughout theentire ambient oxygen range: pO₂=0-160 mm Hg. The probes are based onbright phosphorescent Pt and Pd complexes of porphyrins andsymmetrically π-extended porphyrins (tetrabenzoporphyrins andtetranaphthoporphyrins).

In certain embodiments of the invention, π-extension of the coremacrocycle provides tuning of the spectral parameters of the probes inorder to meet the requirements of a particular imaging application(e.g., oxygen tomography versus planar microscopic imaging).Metalloporphyrins are encapsulated into poly(arylglycine) dendrimers,which fold in aqueous environments and create diffusion barriers foroxygen, making it possible to regulate the sensitivity and the dynamicrange of the method. The periphery of the dendrimers is modified withpoly(ethylene glycol) residues, which enhance the probe's solubility,diminish toxicity, and help prevent interactions of the probes with thebiological environment. The probe's parameters were measured underphysiological conditions, and the probes were shown to be unaffected bythe presence of biomacromolecules.

The performance of the probes was demonstrated in applications,including in vivo microscopy of vascular pO₂ in the rat brain.

As used herein, “DAPIP” shall mean a meso-unsubstituteddiphthalimidoporphyrin.

As used herein, “TAPIP” shall mean a meso-unsubstitutedtetraphthalimidoporphyrin.

As used herein, 1P shall mean “one-photon.”

As used herein, 2P shall mean “two-photon.”

As used herein, 2PA shall mean “two-photon absorption.”

As used herein, 2PLM shall mean “two-photon phosphorescence lifetimemicroscopy”.

I. Principles of Phosphorescence Quenching Method and Requirements of InVivo Oxygen Probes

Phosphorescence quenching relies on the ability of molecular oxygen,which is a triplet molecule in the ground state (O₂X³Σ_(g) ⁻), to reactwith molecules in their excited states, quenching their luminescence.Collisional quenching is much less probable on the time scale of singletexcited states (nanoseconds) than of triplet states (microseconds tomilliseconds), making phosphorescence significantly more sensitive tooxygen than fluorescence. Assuming a large excess of oxygen relative tothe concentration of triplet emitters—a condition typically met inbiological environments—the dependence of the phosphorescence intensityand lifetime on oxygen concentration follows the Stern-Volmerrelationship:

I₀/I=τ₀/τ=1+K_(SV)[O₂],  [Equation 1]

where I and τ are the phosphorescence intensity and the lifetime atoxygen concentration [O₂] and in the absence of oxygen (I₀, τ₀); andK_(SV) is the Stern-Volmer quenching constant.

In practice, using lifetime τ as the analytical signal for [O₂] is moreaccurate, because the lifetime is independent of the probe distributionand of any other chromophores present in the biological system. where τis the phosphorescence lifetime at oxygen concentration at a specificoxygen pressure [pO₂]; τ₀ is the phosphorescence lifetime in the absenceof oxygen [pO₂=0]; and k_(q) is the Stern-Volmer quenching constant.See, Lebedev et al., ACS Appl. Mater. Interfaces 1:1292 (2009); Finikovaet al., ChemPhysChem 9:1673 (2008); Sakad et al., Nat. Methods 7:755(2010); Lecoq et al., Nat. Med. 17:893 (2011); Vinogradov & Wilson, InDesigning Dendrimers, Campagna, Ceroni, Puntoriero, Eds.; Wiley: 2012.

Among the attractive features of the phosphorescence quenching techniqueare its high specificity, submillisecond temporal response, highsensitivity, and relative simplicity of instrumentation. When solubleprobes are used, calibration of the method is absolute in a sense thatthe probes' quenching parameters must be determined only once, and thenthey may be used thereafter for measurements under similar conditions.

It is customary to express oxygen content in the units of pressure (mmHg) rather than concentration (M), since in the majority of biologicalexperiments partial pressure of oxygen (pO₂) is the actually controlledexperimental parameter. At 298 K and the air pressure of 760 mm Hg(oxygen fraction in the air is 21% or 159.6 mm Hg), air-equilibratedaqueous solutions are 252 μM in O₂ (Fogg, et al., “Solubility of gasesin liquids. Wiley, New York, 2001). However, for simplicity the pressureof water vapor above the solution is neglected, although at highertemperatures it will rise and, consequently, the partial pressure ofoxygen (pO₂) will decrease. This assumes that the Henry's law holds inthe physiological range of oxygen concentration: [O₂]=α×pO₂, where α isthe oxygen solubility coefficient (M×mm Hg⁻¹) for the bulk phase.Considering K_(SV)=k₂τ₀, where k₂ is the bimolecular rate constant forthe quenching reaction, Equation 1 can be rewritten as:

1/τ=1/τ₀+(k _(q))(pO₂]  [Equation 2]

where k_(q)=αk₂ and has the units of mm Hg⁻¹s⁻¹. Equation 2 contains twoparameters specific to the molecule of the probe: constant k_(q) andlifetime τ₀. Their interplay defines the sensitivity and the dynamicrange of the method. For analytical purposes, it is desirable that themeasurement parameter, which is the phosphorescence lifetime τ herein,spans the largest possible interval of values throughout the range ofanalyte concentrations, assuring highest possible measurementresolution. To quantify the dynamic range of lifetimes, parameterR=(τ₀−τ_(air))/τ₀ is used, wherein τ₀ (pO₂=0 mm Hg) and τ_(air)(pO₂=159.6 mm Hg) are the maximal and the minimal values of thephosphorescence lifetimes in physiological experiments. Anotherimportant parameter is the signal-to-noise ratio (SNR), which isobviously higher for probes with larger emission quantum yields(assuming the same emission wavelengths).

Pt and Pd porphyrins are typically used as phosphorescent chromophoresfor oxygen measurements in biological systems (vide infra). Tripletlifetimes of Pd porphyrins in deoxygenated solutions at ambienttemperatures are in the range of hundreds of microseconds (abbreviationshave their normal meaning herein, but microseconds are specificallyreferred to “μs” or “μsec” herein), and their phosphorescence quantumyields in the absence of oxygen (φ₀ are typically 0.05-0.1. (Eastwood etal., J. Molecular Spectroscopy 35:359 (1970)). For Pt porphyrins thecorresponding values are tens of microseconds and 0.10-0.25 (Id.; Kim etal., J. Amer. Chem. Soc. 106:4015 (1984)), respectively. Constants k_(q)for “unprotected” metalloporphyrins in aqueous solutions are ˜3,000 mmHg⁻¹s⁻¹. To illustrate how quenching constants k_(q) and lifetimes τ₀affect oxygen measurements, consider two probes, PdP and PtP,representing some arbitrary Pd and Pt porphyrins: τ₀ (PdP)=500 μs,τ_(o)(PtP)=50 μs; φ₀ (PdP)=0.05, φ₀ (PtP)=0.10, where subscript “0”indicates pO₂=0 mm Hg.

When the quenching constant is high, e.g., k_(q)=3,000 mm Hg⁻¹s⁻¹, thelifetime of PdP decreases from τ₀=500 μs to τ_(air)=2.1 μs, resulting ina large dynamic range (R≈0.996). However, between about 20 mm Hg and airsaturation, the lifetime changes by no more than 3% of the total value,between 16 μs and 2.1 μs. Moreover, already at 10 mm Hg. the probe'squantum yield becomes extremely low (φ<0.003) due to the excessivequenching. Consequently, a probe like PdP is useful only in a very lowrange of oxygen concentrations, probably not higher than at pO₂˜5 mm Hg.

Due to its higher quantum yield and shorter τ₀, probe PtP can be used upto about 50 mm Hg (τ=5.9 μs), but above that limit its lifetime alsochanges very weakly, by no more than ˜3 μs. The lifetime dynamic rangeof PtP (R≈0.96) is not very different from that of PdP; but if thequenching constant were to be reduced or decrease, which is inevitableupon binding of the probe to proteins in vivo (vide infra), this probewould become insensitive to changes in oxygenation.

For higher sensitivity, probes with a greater τ₀ are preferred, but onlyif their phosphorescence changes gradually instead of being highlyquenched already at very low oxygen concentrations. Such adjustment ofsensitivity can be achieved by varying constants k_(q). Overall, it isclear that control over the values of the quenching constants and theability to keep them unaltered in measurement environments is the key toaccurate oxygen measurements. All comparative terms, e.g., higher,lower, increased, decreased, enhanced, reduced, faster, slower, etc,assume their accepted standard dictionary meanings herein, preferable ascompared to a stated molecule, probe, compound, or the like.

Phosphorescence quenching by oxygen typically occurs much faster thanthe diffusion of the reactants and formation of encounter complexes; andin the majority of cases, diffusion can be considered the rate-limitingstep for oxygen quenching reaction. (Turro, Modern MolecularPhotochemistry, University Science Books, Sausalito, C A, 1991). Byaltering oxygen diffusion coefficients in the local vicinity of thephosphorescent chromophores, constants k_(q) can be regulated. Suchtuning can be achieved by means of dendritic encapsulation.

Additional requirements for phosphorescent probes for medical imaging ofoxygen include lack of toxicity and preferably excretability from theblood upon completion of imaging. Globular uncharged molecules withmolecular weights up to about 15 kDa are usually excretable by thekidney (Caliceti et al., Advanced Drug Delivery Reviews 55:1261-1277(2003). If a probe satisfies this criterion and remains confined to theintravascular space (does not diffuse out of the blood vessels), it islikely to be removed from the blood by the kidney-mediated dialysis, andthe possibility of long-term toxicity effects can be avoided. Of course,for animal studies, excretability is not as stringent a requirement,whereas confinement to a particular tissue compartment (intravascular,interstitial or intracellular) can be very important. In that case,probes of larger sizes may become advantageous.

This leads to a molecular design comprising a bright phosphorescentchromophore with sufficiently long triplet lifetime τ₀ and a protectivejacket, whose purpose is to constrain oxygen diffusion in the localenvironment of the chromophore. The dendrimer itself must be hydrophobicto permit it to fold (or collapse) in polar solvents, such as water,around the porphyrin. Otherwise, if it were hydrophilic, it would spreadbranches freely and create no effective barrier for oxygen diffusion tothe porphyrin. Importantly, “hydrophobic” does not necessarily meanlipophilic. In other words, a compound that “does not like water” (likethe arylglycine (AG) dendrimers defined below) does not mean that italways “likes” every possible type of organic media. For example, theinventors' arylglycine dendrimers fold both in water and in many organicsolvents, such as, but not limited to tetrahydrofuran (THF), oils,hexanes, and the like. However, in other organic solvents, such asdimethylformamide (DMF) or dimethylsulfoxide (DMSO), they would spreadbranches and would not fold. As a result, such dendrimers would notprovide a barrier for oxygen in DMF and DMSO; but they would do so inwater, THF, oils, alkanes etc.

The periphery of the probe must be hydrophilic and inert in order toprevent interactions with components of the biological system (e.g.,bio-macromolecules, cellular membranes). The overall size of themolecule should be large enough to prevent probe leakage through thevascular walls, but yet small enough to allow removal through thekidney, if excretability is desired. Alternatively, the periphery of theprobe must be hydrophopbic if the measurement is to be conducted inoils, alkanes, etc.

A novelty of the present invention lies in making the folded moleculessoluble. That is accomplished by adding an outer layer to the dendrimer,and it is the outer layer that then takes the probe into solution, evenwhen the dendrimer itself is folded and poorly soluble. Consequently inthe present invention, to dissolve a folded dendrimer in water, ahydrophilic outer layer of polyethyleneglycols (PEG) is added to permitdissolution; but when e.g., oils or long alkyl chains are added to theouter layer, the dendrimer is then soluble in liquid organic compounds.

Overall the problem of keeping the diffusional accessibility of thephosphorescent chromophore to oxygen in biological environments constantthroughout the studied object and the calibration unchanging, is by farthe biggest challenge in the design of phosphorescent oxygen probes.Moreover, this problem is inherent in all methods relying on kinetics ofoxygen diffusion. To overcome this difficulty, design of a molecularoxygen sensor entails construction of a well-defined microenvironmentaround the phosphorescent chromophore in order to isolate it frominteractions with other molecules, except for oxygen. Dendriticencapsulation (Hecht & Fréchet, Angewandte Chemie-International Edition40:74 (2001); Gorman & Smith, J. Amer. Chem. Soc. 122:9342 (2000))arguably provides one of the most straightforward ways to constructmonodisperse, well-defined molecular jackets around luminescentchromophores (Balzani et al., Functional and Hyperbranched BuildingBlocks, Photophysical Properties, Applications in Materials and LifeSciences 228:159 (2003); Ceroni et al., Progress in Polymer Science30:453 (2005)).

II. Phosphorescent Cores of Dendritic Oxygen Probes

The key photophysical properties required for biological oxygen sensingare strong absorption, preferably in the near infra-red region (NIR) ofthe spectrum (near infrared region of tissue is approximately 600 nm to1000 nm, but for excitation purposes from ˜600 or ˜620 to ˜900 or ˜1000nm is appropriate) to minimize the interference by the natural tissuechromophores (e.g., heme containing proteins, carotenoids), and stronglong-lived phosphorescence in solutions at ambient temperatures. Veryfew chromophores possess such properties. Among them, metalloporphyrinsemit from (π-π*) states, and exhibit significantly longer tripletlifetimes (Eastwood et al., J. Mol. Spectroscopy 35:359 (1970)), thuspossessing much higher intrinsic sensitivity to oxygen.

Regular meso-tetraarylated Pt and Pd porphyrins with various peripheralsubstituents can be readily synthesized via, e.g., the Lindsey (Lindseyet al, J. Organic Chem. 52:827 (1987)) or Senge (Senge, Accounts ofChemical Research 38:733 (2005)) methods. Thermodynamic stabilities ofthese complexes are extremely high, (Buchler, Ch. 5 in Porphyrins andMetalloporphyrins, Smith, K. M. Ed., Elsevier, 1975, New York) entirelyprecluding release of free metal ions into biological environments andassociated toxicity. The major drawback of using regular Pt and Pdporphyrins for in vivo applications is that their absorption bands, theso-called Q-bands, are positioned in the visible range (λ_(Q)˜520-530nm; ε˜20,000 M⁻¹cm⁻¹), thus overlapping with absorption of naturallyoccurring chromophores. Still, absorption in the visible region may beuseful in planar wide-field phosphorescence imaging, where less diffusenature of excitation serves to improve spatial resolution. In addition,absorption near 500 nm overlaps with emission of many two-photon (2P)chromophores, which is useful in construction of FRET-enhanced probesfor two-photon oxygen microscopy (vide infra) (Finikova et al.,ChemPhysChem 9:1673 (2008)).

Many porphyrinoids possess near infrared absorption bands; however, veryfew have characteristics appropriate for oxygen sensing (Vinogradov &Wilson, J. Chem. Soc., Perkin Trans. II 103 (1995)). The most usefultoday are derivatives of so-called laterally π-extended (or π-expanded)porphyrins. Lateral π-extension of Pt and Pd porphyrins by annealingtheir pyrrole residues with external aromatic rings results inchromophores with dramatically red-shifted absorption bands and strongroom-temperature phosphorescence (Ibid; Tsvirko et al., Optika iSpektroskopiya (Russ) 34:1094 (1973); Rozhkov et al., InorganicChemistry 42:4253 (2003)). Spectra of Pt analogs are very similar inshape, but are blue-shifted by ˜10-15 nm as compared to those of Pdcounterparts.

An important feature of all porphyrin-based probes is their very largeseparation between the absorption and phosphorescence, achievable viaexcitation at the Soret bands (e.g., 9329 cm⁻¹ for Pd porphyrins).Efficient S₂→S₁ internal conversion, (Tripathy et al., J. Phys. Chem. A112:5824 (2008)) combined with extremely high extinction coefficients ofS₀-S₂ transitions (˜3×10⁵ M⁻¹cm⁻¹) makes this pathway superior to thedirect S₀-S₁ excitation in those cases when near UV radiation can besustained by the biological object.

Until recently, synthesis of π-extended porphyrins presented achallenging problem. All synthetic methods were based on hightemperature condensations between phthalimide (or naphthalimide) andarylacetic acids, or similar donors of benzo- and phenyl-groups(Kopranenkov et al., J. Gen. Chem. (Russ) 51:2165 (1981); Edwards etal., J. Amer. Chem. Soc. 98:7638 (1976); Ichimura et al., InorganicaChimica Acta 176:31 (1990)). The harsh conditions of condensation(melting at 300-400° C.) allowed for only a few inert substituents, suchas alkyl groups or halogens (Kopranenkov et al., KhimiyaGeterotsiklicheskikh Soedinenii 773 (1988)) to be introduced into theporphyrin macrocycle. In addition, low yields and complex, inseparablemixtures of products made this approach impractical.

The newly emerged approaches to π-extended porphyrins rely on theBarton-Zard methodology (Barton et al., Tetrahedron 46:7587 (1990)) forsynthesis of porphyrinogenic pyrroles, which give rise to precursorporphyrins requiring final aromatization. Two methods have beendeveloped into practical synthetic schemes: one making use ofretro-Diels-Alder reaction (Ito et al., Chemical Communications 1661(1998); Ito et al., Chem. Comm. 893 (2000)), another relying on simpleand efficient oxidative aromatization strategy (Finikova et al., Chem.Commun. 261 (2001); Finikova et al., J. Organ. Chem. 69:522 (2004)). Thelatter method is being used today to synthesize tetrabenzo- andtetranapthoporphyrins for construction of phosphorescent oxygen probes,as well as in several other applications.

Although meso-tetraarylated π-extended porphyrins are most common,primarily because of the solubility considerations, meso-unsubstitutedtetrabenzoporphyrins (TBP's) and tetranaphthoporphyrins (TNP's), whichin fact have higher phosphorescence quantum yields and longer lifetimes,can also be synthesized via the oxidative aromatization method (Finikovaet al., J. Org. Chem. 70:9562 (2005)). Furthermore, recent introductionof 4,7-dihydroisoindole (Filatov et al., Europ. J. Org. Chem. 3468(2007)) paved a practical route to 5,15-diaryl-TBPs (Filatov, et al., J.Org. Chem. 73:4175 (2008)). The latter porphyrins are especiallyattractive for construction of oxygen probes, as they combine higheremissivity (Lebedev et al., J. Phys. Chem. A 112:7723 (2008)) with thepossibility of attaching dendrons to the anchor points in the meso-arylrings, which is lacking in meso-unsubstituted porphyrins. Notably,examples of porphyrin-dendrimers based on 5,15-diarylporphyrins are wellknown (Dandliker et al., Angewandte Chemie-International (Edition inEnglish) 33:1739 (1994); Dandliker et al., AngewandteChemie-International (Edition in English) 34:2725 (1996)).

III. Polyglutamic Pd Porphyrin-Dendrimers

Polyglutamic Pd porphyrin-dendrimers made up the first generation ofdendritic oxygen probes (Vanderkooi et al., J. Biol. Chem. 262:5476(1987); Vinogradov & Wilson J. Chem. Soc., Perkin Trans. 2:103 (1995);Papkovsky et al., J. Fluoresc. 15:569 (2005)). The compounds aredescribed by the general formula, e.g., PdP-(Glu^(n)OH)₁, if firstgeneration, where n=1-4 (dendrimer generation) and Glu^(n)OH is theglutamic layer. Similar abbreviations to designate porphyrin dendrimersof different types and generations, terminated by different groups areused herein. But the polyglutamic Pd porphyrin-dendrimers requiredprebinding to macromolecular carriers (e.g., albumin) in order toenhance their aqueous solubility and bring their quenching parameters(τ₀ and k_(q), Eq 1) into the range compatible with physiological oxygenconcentrations (see Vinogradov & Wilson, In Designing Dendrimers, chap14, Campagna, Ceroni, Puntoriero, Eds.; Wiley: 2012) for discussion).However, foreign albumin was a potential source of toxicity andimmunogenic responses.

Introduction of polyglutamic dendritic porphyrins Vinogradov et al.,Chem. Eur. J. 5:1338 (1999); Vinogradov et al., Adv. Exp. Med. Biol.428:657 (1997); Rietveld et al., Tetrahedron 59:3821 (2003), which weregeneration 2 (gen 2) polyglutamic PD prophyrin dendrimers, known asOxyphors R2 and G2 (Dunphy et al., Anal. Biochem. 310:191 (2002)),offered a partial solution to this problem, and they have been used inmany biological studies over the years, wherein dendritic encapsulationwas needed to protect the triplet states of Pd porphyrins from oxygenquenching. Polyglutamic probes have high intrinsic aqueous solubilityand can be introduced into the blood directly without prebinding toalbumin. When in the blood, they form complexes with endogenous albumin,and these complexes serve as ultimate oxygen sensors. But as a result,use of Oxyphors R2 and G2 is limited to albumin-rich environments, suchas blood plasma (>2% in albumin by weight). But even in suchenvironments, incomplete binding to albumin, which can be easilyencountered at higher probe concentrations (e.g., above ˜10⁻⁵ M), maylead to heterogeneity of the probe signal, skewing the measurement.(Vinogradov et al., Applied Spectroscopy 54:849 (2000)). Notably,combinations of polyglutamic probes with polymeric nanoparticle-basedcarriers have been recently introduced (e.g., Lee et al., Anal. Chem.82:8446 (2010)) which alleviate the necessity of albumin binding andallow oxygen measurements in cultured cells.

Thus, the drawbacks associated with use of dendritic polyglutamicporphyrins clearly show that probes with albumin-independentphosphorescence lifetimes and oxygen quenching constants would greatlysimplify data analysis and broaden applicability of the method (e.g.,make it possible to take measurements in albumin-free environments).Although polyglutamic dendrimers were limited in use, experiments showedthat composition of the dendritic matrix is at least as important forshielding as is the dendrimer size itself, leading to studies of theinterplay between the dendrimer composition, size and encapsulatingefficiency, as measured by oxygen diffusion and quenching ofphosphorescence.

IV. Influence of Size and Composition of Dendritic Matrix on OxygenShielding Efficiency

In order to determine which dendrimers provide optimal attenuation ofoxygen quenching of porphyrin phosphorescence a study was performed(Rozhkov et al., Macromolecules 35:1991 (2002)) which involved threetypes of porphyrin-dendrimers: Fréchet-type poly(aryl ethers) (Hawker etal., J. Chem. Soc. Perkin Trans. I 1287 (1993)), Newkome-type poly(etheramides) (Newkome et al., Macromolecules 24:1443 (1991); Newkome et al.,Synlett 53 (1992)), and polyglutamates (Vinogradov et al., Chemistry-AEuropean J. 5:1338 (1999)).

Pd-meta-octahydroxyphenylporphyrin andPd-meta-octacarboxyphenyl-porphyrin, compatible with either Williamson(Fr) or peptide (Nw, Glu) chemistries, were used as phosphorescentcores. The studies were performed in dimethylformamide (DMF),tetrahydrofuran (THF) and water. To insure appropriate aqueoussolubility, termini of the dendrimers were modified witholigoethyleneglycol residues, e.g., PEG (polyethylene glycol) whichrendered uncharged molecules soluble both in organic solvents and inwater.

In organic solvents, decrease in the quenching rates was found to beconsistent with increase in the dendrimer size, and these changes wererather insignificant. As expected, bulkier poly(ether amides) exhibit astronger effect, but still the decrease in the quenching was only about75% for G2 dendrimer, whose weight is more than 12 kDa, that is morethan 12 times that of the core porphyrin. Notably, in the case ofpolyglutamic derivatives of PdTCPP, dendrons were attached to thepara-positions on the meso-aryl rings of the porphyrin, extending out ofthe macrocycle instead of covering it from above and below, and thus theshield effect was much weaker for the same size dendrons. A much largerdifference in quenching rates was observed in water.

The practical determination was that dendrimers constructed fromaromatic building blocks potentially have much higher shieldingefficiency in aqueous environments than do other types of dendrimers. Inaddition, it was determined that in order to prevent interaction ofdendrimers with biological macromolecules, their periphery should bemodified with PEG residues (vide infra), referred to as “peripheralPEGylation” of the dendrimers.

Other references relating to dendritic macromolocules and their methodsof production can be found in U.S. Pat. Nos. 5,418,301; 4,568,737;5,393,795; 5,256,193; 5,393,797; 5,393,795; 5,393,797; 5,098,475;5,041,516 and 4,568,737, the entire disclosures of which areincorporated herein by reference. The phosphors employed in, forexample, U.S. Pat. No. 5,837,865 (the '865 patent), and by Vinogradovand Wilson, J. Chem. Soc., Perkin trans. 2:103-111 (1995), werepreferably of the general formula described therein, wherein R₁ washydrogen (H) or substituted or unsubstituted aryl. R₂ and R₃ wereindependently hydrogen or are linked together to form substituted orunsubstituted aryl. When R₂ and R₃ were linked together to form an arylsystem, the aryl system was necessarily in a fused relationship to therespective pyrrole substrate. M was H₂ or preferably a metal selectedfrom the group consisting of Lu, Pd, Pt, Zn, Al, Sn, Y La, andderivatives thereof, with Pd, Pt and Lu being most preferred.

Accordingly, the prior art formulations were all based onmeso-tetraaryl-substituted metalloporphyrins, but the inventors havedetermined that non-planar deformation of the porphyrin macrocycleresulting from tetra-aryl substitution led to a loss of phosphorescenceemission. Thus, the resulting dendritic oxygen probes were effective,but limited for their intended purpose.

V. Protected Dendritic Probes: Oxyphors G4 and R4

Subsequently however, the inventors identified a general approach toprotected molecular oxygen probes, which do not require albumin or anyother supporting macromolecular carriers or nanocompositions (See,Lebedev et al., ACS Appl. Mater. Interfaces 1:1292 (2009); Finikova etal. ChemPhysChem 9:1673 (2008); Ceroni, et al., Photochem. Photobiol.Sci. 10:1056 (2011)). In these probes, phosphorescent metalloporphyrinsare encapsulated inside hydrophobic dendrimers, which form protectingshells, enveloping the chromophores, controlling oxygen diffusion to theexcited triplet states, and enabling control over the sensitivity of theapproach. Peripheral PEGylation of the dendrimers ensured high aqueoussolubility and prevented interactions of the probes with biologicalmacromolecules.

Synthesis and detailed characterization of two new probes, Oxyphors R4and G4, derived from phosphorescentPd-meso-tetra-(3,5-dicarboxyphenyl)-porphyrin (PdP) orPd-meso-tetra-(3,5-dicarboxyphenyl)-tetrabenzoporphyrin (PdTBP),respectively, as reported by Esipova et al, Anal. Chem. 83:8756(published on-line Oct. 11, 2011), herein incorporated by reference.These probes are built according to the above-referenced general scheme,and possess features common for protected dendritic probes, i.e.,hydrophobic dendritic encapsulation of phosphorescent metalloporphyrinsand hydrophilic PEGylated periphery. The new Oxyphors R4 and G4 arehighly soluble in aqueous environments and do not permeate biologicalmembranes. The probes were calibrated under physiological conditions (pH6.4-7.8) and temperatures (22-38° C.), showing high stability,reproducibility of signals, and lack of interactions with biologicalsolutes were intended for wide use in biological research. However, theprobes' structural elements are further optimized for improved yield ofsynthesis, 1-2 times higher monodispersity of pure monodispersedendritic oxygen probes, lack of aggregation in aqueous solutions, andoverall better chemical stability.

As disclosed above a byproduct of the biological oxygen measurements byphosphorescence quenching using exogenous phosphorescent probesintroduced directly into the medium of interest (e.g., blood orinterstitial fluid) is singlet, a highly reactive species capable ofdamaging biological tissue. Consequently, because potential probephototoxicity is a concern for biological applications studies comparedthe ability of polyethyleneglycol (PEG)-coated Pd tetrabenzoporphyrin(PdTBP)-based dendritic nanoprobes of three successive generations tosensitize singlet oxygen. As a result, it was demonstrated that the sizeof the dendrimer has practically no effect on the singlet oxygensensitization efficiency in spite of the strong attenuation of thetriplet quenching rate with an increase in the dendrimer generation.

Nevertheless, in spite of their ability to sensitize singlet oxygen, thephosphorescent probes were found to be non-phototoxic when compared withthe commonly used photodynamic drug Photofrin in a standardcell-survival assay. (Ceroni et al., Photochem. Photobiol. Sci.10:1056-1065 (2011)). The lack of phototoxicity is presumably due to theinability of PEGylated probes to associate with cell surfaces and/orpenetrate cellular membranes. In contrast, conventional photosensitizersbind to cell components and act by generating singlet oxygen inside orin the immediate vicinity of cellular organelles. Therefore, PEGylateddendritic probes are safe to use for tissue oxygen measurements as longas the light doses are less than or equal to those commonly employed inphotodynamic therapy.

One of the probes, Oxyphor G4 was applied to imaging of oxygendistributions in tumors, both intravascularly and in the interstitialspace. The probe allowed dynamic visualization of the tissue pO₂ levelsin the tumor and in the surrounding muscle, as oxygenation responded tochanges in the depth of anesthesia. The phosphorescence from the probealso could be detected in trans-illumination geometry, i.e., through thewhole body of the animal, thus demonstrating feasibility of in vivo fullbody oxygen tomography.

VI. Effect of the Dendrimer Outer Layer

Although it is critically important that quenching properties ofphosphorescent probes do not change in the presence of blood plasmaproteins and other biological macromolecules, quenching constants of allcarboxylate-terminated porphyrin-dendrimers were found to be highlysensitive to albumin. For example, k_(q) of gen 2 Fréchet-type porphyrindendrimer in albumin solution (2% by mass) is five times lower (30 mmHg⁻¹ s⁻¹) than that in the albumin-free aqueous buffer (151 mm Hg⁻¹ s⁻¹)(Gorman et al., J. Amer. Chem. Soc. 122:9342 (2000)). To the contrary,however, quenching of porphyrin-dendrimers modified witholigoethyleneglycol residues (PEG350, Av. MW=350) appeared to beunaffected by serum proteins. Thus, k_(q) of the PEGylated analogue ofgen 2 Fréchet-type porphyrin dendrimer was found to have the same value(130 mm Hg⁻¹s⁻¹) in the presence and in the absence of albumin (Estradaet al., Optics Letters 33:1038 (2008)).

Thus, it was important to distinguish between the effect of hydrophobicdendrons and the effect of external PEG residues. While the latter alsocontribute to the protection from quenching, especially at earlydendritic generations, the net effect of PEG is very small, as notedabove, compared to the shielding by dendritic branches. Moreover, thePEG effect rapidly levels off with extension of linear chains. It wasshown that external PEG chains as large as 300,000 Da have practicallyno effect on oxygen quenching constants of these molecules. Moreover, inaddition to being able to prevent interactions of dendrimers withproteins, peripheral PEG groups also strongly affect biodistribution andrate of excretion of dendritic macromolecules (Newkome, supra, 1992).

VII. Aryl-Glycine Dendrimers and Fully Protected Phosphorescent Probes

Among known dendrimers with aromatic backbones, dendritic arylamideswere very attractive because of their high chemical stability, low costand effective protocols available for their assembly. In particular,arylamide dendrimers based on 5-aminoisophthalic acid (5-AIPA) wereselected to make the focal functionality more reactive and tosimultaneously increase the flexibility of the dendritic backbone byextending the amino-end of the 5-AIPA molecule by adding a flexiblefragment, terminated with an aliphatic amine. This possibility wasexplored by constructing aryl-glycine (AG) dendrons and dendrimers(Vinogradov, Organic Letters 7:1761 (2005)). The following abbreviationsare used to designate aryl-glycine dendrons and dendrimers. Dendronswere designated X-AGnR, where AG denotes the dendritic aryl-glycineskeleton, n is the dendrimer generation number, X is the focalfunctionality and R is the terminal group. Dendrimers were designatedC-(AGnR)m, where C denotes the dendrimer core, AG denotes the dendriticaryl-glycine skeleton, n is the generation number, R is the terminalgroup and m is the number of dendritic wedges attached to the core.

The developed synthesis of AG dendrons relies on the classic Fischer'shaloacyl halide method (Miller et al., Chem. Mater. 2:346 (1990)) forthe synthesis of building blocks. Synthesis of AG-dendrons in principlealso can be implemented using the Fischer's method, however, a morerobust scheme is based on a coupling-deprotection sequence employing thepeptide-coupling reactions of Lebedev et al., ACS Applied Materials &Interfaces 1:1292 (2009)). See also synthesis and peptide-couplingreactions described in greater detail in Vinogradov & Wilson, InDesigning Dendrimers, chap 14, Campagna, Ceroni, Puntoriero, Eds.;Wiley: 2012).

Each step of the sequence was carefully optimized in order to insuremaximal yields and monodispersity of the probes. Porphyrins modifiedwith AG¹ and AG² (aryl-glycine, first and second generation) dendronscould be isolated in high purity, but modification of porphyrins withAG³-dendrons (third generation) proved extremely challenging, alwaysyielding mixtures of dendrimers with six, seven and eight dendriticbranches. The peripheral ester groups were hydrolyzed using a specialtwo-step procedure (Lebedev et al., supra, 2009), which in paralleldestroys unreacted dendrons, making it possible to entirely avoidchromatographic purifications. The resulting peripheral carboxyl groupson the dendrimers are esterified with PEG residues of the desiredlength, and simple re-precipitation from THF upon addition of diethylether produced pure PEGylated dendrimers. The magnitude of theattenuation effect is similar to that of poly(aryl ether) dendrimers. Asa result quenching constants and lifetimes of gen 2 Pdporphyrin-dendrimers are well suited for biological oxygen measurements.These probes exhibit good dynamic range and excellent sensitivity. Forexample, in the case of PdP-based gen 2 dendrimer the relativesensitivity coefficient R (vide supra) is as high as 0.9. Moreover,PEG-coated AG porphyrin-dendrimers exhibited a property that theiroxygen quenching constants kq and lifetimes τ₀ are completelyinsensitive to the presence of biological macromolecules.

VIII. Improvements in the “Next Generation” of Oxygen Sensors:Meso-Unsubstituted Porphyrin Embodiments.

A) Increased Emission Output (Quantum Yield) of PhosphorescentPorphyrin-Based Probes.

Systematic structure/property comparisons between differentlysubstituted porphyrins, have demonstrated that dendritic oxygen probescan be constructed using Pd meso-unsubstituted.

Increase in the phosphorescence quantum yield is an important objective.Pd meso-tetraarylporphyrins have their quantum yields at zero-oxygen inthe range of 0.02-0.08 and triplet lifetimes of 300-700 μs, depending onthe type of π-extension, i.e., tetrabenzo-extension ortetranaphtho-extension with respect to the basic 18-tetrapyrrolicmacrocycle (Rozhkov et al., Inorg. Chem. 42:4253-4255 (2003); Rogers etal., J. Physical Chem. A 107:11331-11339 (2003)). Analogous Ptporphyrins have the quantum yields of 0.10-0.20, when measured bycurrently approved measurement techniques, and their lifetimes are 30-60μs.

The inventors' detailed photophysical, structural and computationalstudies were conducted to delineate the interplay between radiative andnon-radiative triplet deactivation processes in Pd porphyrins andtetrabenzoporphyrins (see Lebedev et al., J. Physical Chem. A,112:7723-77336 (2008)). In fact, non-planar distortions of the porphyrinmacrocycle were shown to diminish the triplet emission yield of PdTBP'scompeting non-radiative triplet-to-singlet ground state transitions,i.e., intersystem crossing. This is because the lifetime of the PdTBPtriplet state in the absence of oxygen increases with dendriticgeneration, thus compensating for the concomitant decrease in the rateof quenching. In fact, non-planar distortions of the porphyrinmacrocycle were shown to diminish the triplet emission yield of PdTBP'sby increasing the rate of T₁→S₀, referring to the internal,non-radiative quenching of the triplet state (T₁) (returning it to theground state (S₀). Increase in this non-radiative pathway decreasesphosphorescence emission. However, this effect is considerably lesspronounced than in regular non-extended porphyrins. Photophysicalmeasurements show that non-planar deformations have deleterious effecton the porphyrin triplet emissivity. Therefore, even highly saddledtetraaryl-TBP's, which are used as cores in current probes, are able tophosphoresce.

It was also determined by the inventors' studies that Pd and Pt TBP'swithout meso-substituents (Finikova et al., J. Org. Chem. 70:9562-9572(2005A)) have much higher emission quantum yields than the correspondingtetraarylporphyrins. In the case of meso-unsubstituted PtTBP's,phosphorescence quantum yields reach as high as 0.5 at room temperature,by currently accepted measuring techniques. The same trends arecharacteristic of TNP's.

Based on these findings, Pt and Pd meso-unsubstituted TBP's and TNP'sare used for construction of certain embodiments of the new generationprobes of the present invention.

In one embodiment of the present invention, the porphyrinogens areassembled using the 2+2 (Filatov et al., “Synthesis of5,15-diaryltetrabenzoporphyrins,” J. Org. Chem. 73:4175. (2008)) andoxidized by DDQ (dichlorodicyano-1,4-benzoquinone) into the targetprecursor tetracyclo-hexenoporphyrin (TCHP) (Ibid). After insertion ofPd or P), the TCHP is oxidized, also by DDQ, into target TBP's (Finikovaet al., “Novel versatile synthesis of substituted tetrabenzoporphyrins,”J. Org. Chem. 69:522 (2004)).

Synthesis of meso-unsubstituted TBP's and TNP's with substituents in thefused rings has been previously developed (Finikova et al., “Synthesisand luminescence of soluble meso-unsubstituted tetrabenzo- andtetranaphtho [2,3] porphyrins,” J. Org. Chem. 70:9562 (2005)). However,these positions are hindered sterically, especially with respect to theattachment of bulky dendritic ligands, required for “protection” of theporphyrin core from excessive oxygen quenching.

In an embodiment of the invention, the scheme depicted in FIG. 1 is usedto synthesize meso-unsubstituted TBP's with appropriately positionedanchor groups as disclosed, with M being selected from H₂, Zn, Ni, CU,Pd, Pt, Ru, Au, Os, Ir, La. Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er,Tm, Yb, and Lu. The scheme shows two routes, sharing maleimide as acommon precursor. One route was based on the modified Barton-Zardsynthesis of sulfolenolpyrrole (a method of pyrrole synthesis by acyclization reaction between a nitroalkene and α-isocyanoacetate underbase conditions) developed previously by Vicente et al., TetrahedronLetters 38:3639-3642 (1997). A second route was also feasible; however,conditions of the Barton-Zard reaction (Barton, Zard, “A new synthesisof pyrroles from nitroalkenes,” J. Chem. Soc. 1098 (1985)), needed to beadjusted for each specific case, as its outcome is likely to be affectedby the presence of aromatic imide group. The stability of this imidedepends on the substituents R and their positions (given thatelectron-withdrawing substituents in 3, 5-positions will de-stabilizethe imide with respect to hydrolytic cleavage). Accordingly, to insurebetter protection of meso-unsubstituted porphyrins already at earlydendritic generations in at least one embodiment, 2,6-substituents wereused, which direct the dendrons under and above the porphyrin plane.

In one embodiment of the invention, the TAPIP and DAMP porphyrinsdepicted in FIG. 2 are the products of the synthesis schemes shown inFIG. 3 and FIG. 4, with M being selected from H₂, Zn, Ni, CU, Pd, Pt,Ru, Au, Os, Ir, La. Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb,and Lu.

FIG. 3 depicts TAPIP synthesis, which starts with the Barton-Zard-typereaction of sulfolene 1, bearing an electro-withdrawing group X (e.g.NO₂ of SO₂Ar), with isocyanoacetate to give sulfolenolpyrrole ester 4.Although FIG. 3 depicts X as being NO2, the invention is not so limited,and with the benefit of the present disclosure, one skilled in the artwould be enabled to begin the synthesis with a sulfolene having anyelectro-withdrawing group at this position. In parallel maleimide 3 issynthesized from maleic anhydride 2 and amine R—NH₂, where group R is anaryl or an akyl, and is selected from the group comprising methyl,ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, decyl, dodecyl,phenyl, tolyl, xylyl, hydroxyphenyl, dihydroxyphenyl, aminophenyl,sulfophenyl, and bromophenyl. In a preferred embodiment, as depicted inFIG. 2, the groups R are 2,6-disubstituted aryl groups, as these areadvantageous for their high solubility. Because of steric hindrancecaused by two substituents R₂, the 2,6-disubstituted aryl group rotatesout of plane of the pyrrole ring in sulfolenolpyrrole 4 as well as inall subsequent structures. This out-of-plane geometry preventsaggregation and increases solubility of the target porphyrins.

Diels-Alder reaction of sulfolenopyrrole ester 4 and maleimide 3generates cyclohexenopyrrole ester 5, which upon cleavage of estergroups gives pyrrole 6. Pyrrole 6 is then introduced into the reactionwith formaldehyde in the presence of an acidic catalyst, followed byoxidation, generating tetracyclohexenoporphyrin 7. In a preferredembodiment, the acidic catalyst is trifluoroacetic acid. This porphyrinmay be metallated using a variety of metal salts, with salts containingmetal M selected from Zn, Ni, Cu, Pd, Pt, Ru, Au, Os, Ir, La, Ce, Pr,Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu being particularlyuseful for gaining useful luminescent and photodynamic properties. Theresulting metalloporphyrin is oxidized into the metallo-TAPIP (9), usingoxidants such as dichlorodicyanobenzoquinone (DDQ) or its analogues. Inan alternative pathway, a free-base version of tetracyclohexenoporphyrin7 can be oxidized into TAPIP free-base (9a) and then metallated with ametallic salt having metal M, wherein metal M is selected from the groupcomprising Zn, Ni, Cu, Pd, Pt, Ru, Au, Os, Ir, La, Ce, Pr, Nd, Pm, Sm,Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu, to produce a metalloporphyrin.

FIG. 4 depicts a similar synthesis scheme to be applied for the purposeof synthesizing DAPIP rather than TAPIP. The first steps of thissynthesis are the same as those in the synthesis of TAPIP, and areincorporated herein by reference; however after assembling pyrrole ester5, instead of ester cleavage/decarboxylation, pyrrole ester 5 isintroduced into reaction with itself to give dipyrromethane-diester 10.Dipyrromethane-diester 10 is then de-esterified, decarboxylated andcondensed with diformyldipyrromethane 11. Syntheses of dipyrromethanes11 are well-developed and known from the literature, and substituents R1and R2 can vary between different alkyl and aryl groups. In a preferredembodiment, where R1=R2=H, dipyrromethane 11 is unsubstituteddiformyl-dipyrromethane, which can be generated in bulk quantities byreaction of pyrrole with formaldehyde in excess pyrrole used as asolvent, followed by formylation into α-positions. Importantly, groupsCl are formyl groups, synthons of the same, or any groups exhibiting thesame formal reactivity as formyl groups and yielding the same products;in any case the groups Cl yield formyl groups in situ during theacid-catalyzed reaction with diformyldipyrromethane 11.

In one embodiment, the group R of amines R—NH2 are 2,6-disubstitutedaryl groups, as these are advantageous for their high solubility.

The subsequent reactions—metallation followed by oxidation or viceversa—exactly replicate those in the synthesis of TAPIP depicted in FIG.3. Just as with TAPIP, there is an alternative pathway in which afree-base version of dicyclohexenoporphyrin is oxidized to yield DAPIPfree-base. This DAPIP free-base is then, in turn, metallated with ametallic salt having metal M, wherein metal M is selected from the groupcomprising Zn, Ni, Cu, Pd, Pt, Ru, Au, Os, Ir, La, Ce, Pr, Nd, Pm, Sm,Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu, to produce a metalloporphyrin.

B) Luminescence of Meso-Unsubstituted Phthalimidoporphyrins.

Metal complexes of TAPIP and DAPIP with 2,6-disubstituted aryl groups Rare well-soluble in organic solvents. These metalloporphyrins havestrong absorption in the visible/near infrared regions of the spectrum.Pt and Pd complexes phosphoresce at ambient temperature with very highquantum yields (from 0.4 to 0.45 at 22 degrees Celsius). Thephosphorescence quantum yields reach 0.45 in organic solvents, such asdimethylformamide, at 22 degrees Celsius—a rare and useful property forapplications. Zn and other lighter metal complexes fluorescence. FIGS.7A-7H depict the absorption and emission spectra, whetherphosphorescence or fluorescence, of Zn, Pd and Pt complexes of DAPIP andTAPIP: FIG. 5A shows the absorption spectra of ZnTAPIP; FIG. 5B showsthe fluorescence of ZnTAPIP; FIG. 5C shows the absorption spectra ofPdTAPIP; FIG. 5D shows the phosphorescence of PdTAPIP; FIG. 5E shows theabsorption spectra of PtTAPIP; FIG. 5F shows the phosphorescence ofPtTAPIP; FIG. 5G shows the absorption of PtDAPIP: FIG. 5H shows thephosphorescence of PtDAPIP.

Bright phosphorescence makes Pt and Pd complexes of DAMP and TAPIP veryvaluable as core chromophores for oxygen sensing based onphosphorescence quenching.

C) Improved Procedure for Dendrimerization of Porphyrins.

Embodiments of the invention have shown that attachment of extensionlinkers to the anchor groups on either meso-aryl rings or directly onporphyrin macrocycle greatly facilitates coupling of the protectingdendrons. Specifically, extension of carboxylic groups withγ-aminobutyrate linkages (“extension arms”) permitted high yieldsynthesis of monodisperse dendritic oxygen probes or improvedsensitivity and higher purity (See, basic photophysical studies as afoundation for determining the significantly higher phosphorescencequantum yields of the 5,15-diaryl-substituted π-extended porphyrins, ascompared with tetraarylporphyrins by Esipova et al., “Two new“protected” oxyphors for biological oximetry: properties and applicationin tumor imaging,” Analytical Chem. 83:8756 (published on-line Oct. 11,2011).

Dendrimerization of phosphorescent porphyrins includes hydrolysis of theperipheral ester groups on the porphyrin, attachment of dendrons,hydrolysis of their peripheral esters on the dendrons and PEGylation ofthe resulting free carboxyl groups. See, FIG. 1. These methods have beendeveloped by the inventors and described in their previous patents citedabove, and incorporated herein by reference. One critical improvement,however, that was only recently established, relates to the yield andpurity-limiting step in the synthesis—that is, attachment of the bulkydendritic substituents to the porphyrin core. This step typicallyproceeds via one of the standard peptide coupling reactions, or anyother suitable chemistry. Yet the steric hindrance imposed by largefolded dendritic groups impedes the reaction. However, if the anchorgroups are extended by flexible linkers, bulky substrate react slowlybecause of unfavorable steric reasons, and thus the actual reactionsites are placed at a larger distance from one-another, they become muchmore accessible to nucleophilic attack. Consequently, the resultingreaction occurs with higher rate, i.e. complete dendrimerization isreached overnight (approx. 24 hours), as opposed to 7 days in the caseof porphyrins without extension arms, and provides cleanocta-dendrimerized porphyrin, as opposed to mixture of hexa-, hepta- andocta-dendrimerized porphyrins (imperfect contaminating dendrimers) thatwere obtained in the case of porphyrins without extension arms.

The overall scheme of the synthesis is shown in FIG. 7 for the case of5,15-diaryl-p-extended porphyrins (vide supra), with M being selectedfrom H₂, Zn, Ni, CU, Pd, Pt, Ru, Au, Os, Ir, La. Ce, Pr, Nd, Pm, Sm, Eu,Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu. The extension step is the first stepin the reaction sequence, and the addition of AG3 elements (thirdgeneration (Gen 3)) arylglycine dendrimers.

D) Increase in the Excitation Cross-Sections of the Probe ViaIntramolecular Fluorescence Resonance Energy Transfer (FRET).

One approach to enhancing production of excitement cross-sections inmetalloporphyrins without directly altering their electronic propertiesinvolves harvesting the excitation energy by an electronically separateantenna and to pass it onto the phosphorescent core via intramolecularFörster-type resonance energy transfer (FRET) (See, Briñas et al., J.Amer. Chem. Soc. 127:11851 (2005)). In such an antenna-core systemconstructed around a dendrimer, the latter regulates the rate of oxygendiffusion to the core, just as in the regular dendritic probes (seeabove), while the dendrimer termini control the probe bio-distribution.

Resonance energy transfer is the radiationless transmission of an energyquantum from its site of absorption to the site of its utilization inthe molecule, or system of molecules, by resonance interaction betweenchromophores, over distances considerably greater than interatomicdistance, without conversion to thermal energy, and without the donorand acceptor coming into kinetic collision. The donor is the marker thatinitially absorbs the energy, and the acceptor is the chromophore towhich the energy is subsequently transferred. (from Van Der Meer, Coker,and Chen, Resonance Energy Transfer Theory and Data, VCH, New York,1994).

Förster (Fluorescence) resonance energy transfer (FRET), resonanceenergy transfer (RET) or electronic energy transfer (EET), is amechanism describing energy transfer between two chromophores. [1] Adonor chromophore, initially in its electronic excited state, maytransfer energy to an acceptor chromophore through nonradiativedipole-dipole coupling. [2] The efficiency of this energy transfer isinversely proportional to the sixth power of the distance between donorand acceptor making FRET extremely sensitive to small distances. [3]Measurements of FRET efficiency can be used to determine if twofluorophores are within a certain distance of each other. [4] Suchmeasurements are used as a research tool in fields including biology andchemistry. When both chromophores are fluorescent, the term“fluorescence resonance energy transfer” is often preferred, althoughthe energy is not actually transferred by fluorescence. In order toavoid an erroneous interpretation of the phenomenon that is always anonradiative transfer of energy (even when occurring between twofluorescent chromophores.

FRET is analogous to near field communication, in that the radius ofinteraction is much smaller than the wavelength of light emitted. In thenear field region, the excited chromophore emits a virtual photon thatis instantly absorbed by a receiving chromophore. These virtual photonsare undetectable, since their existence violates the conservation ofenergy and momentum, and hence FRET is known as a radiationlessmechanism. Quantum electrodynamical calculations have been used todetermine that radiationless (FRET) and radiative energy transfer arethe short- and long-range asymptotes of a single unified mechanism.Jablonski diagrams illustrate the electronic states of a molecule andthe transitions between them. The states are arranged vertically byenergy and grouped horizontally by spin multiplicity (Jablonski,Aleksander “Efficiency of Anti-Stokes Fluorescence in Dyes” Nature131:839-840 (1933)).

1) Theoretical Basis of FRET:

When the donor probe is a fluorescent molecule, and when light excitesthe fluorophore at an appropriate wavelength (250-500 nm), its electronsjump from the ground state (S) to a higher vibrational level (S, S₀, S₁,etc). Within picoseconds these electrons decay to the lowest of thesevibrational levels (S) and then decay more slowly (nsec) to one of the Sstates and a photon of light is emitted whose wavelength is longer thanthat of the exciting wavelength. The acceptor probe can be fluorescentor non-fluorescent.

The FRET efficiency depends on many physical parameters that can begrouped as follows:

-   -   The donor probe must have a high quantum yield.    -   The spectral overlap of the donor emission spectrum and the        acceptor absorption spectrum. The emission spectrum of the donor        probe must overlap considerably the absorption spectrum of the        acceptor probe (overlap integral).    -   There is an appropriate alignment of the absorption and emission        moments and their separation vector (embodied in kappa square).    -   The distance between the donor and the acceptor. The donor and        acceptor must be within 1±0.5×r_(o) from each other, when        r=donor-to-acceptor separation distance.    -   The relative orientation of the donor emission dipole moment and        the acceptor absorption dipole moment.

Accordingly, FRET efficiency relates to the quantum yield and thefluorescence lifetime of the donor molecule as follows (See, e.g.,Majoul et al. (2006). “Practical Fluorescence Resonance Energy Transferor Molecular Nanobioscopy of Living Cells.” In Pawley, Handbook OfBiological Confocal Microscopy (3rd ed.). New York, N.Y.: Springer. pp.788-808). In fluorescence microscopy, fluorescence confocal laserscanning microscopy, as well as in molecular biology, FRET is a usefultool to quantify molecular dynamics in biophysics and biochemistry, suchas protein-protein interactions, protein—DNA interactions, and proteinconformational changes. For monitoring the complex formation between twomolecules, one is labeled with a donor, and the other with an acceptor.The FRET efficiency is measured and used to identify interactionsbetween the labeled complexes. After excitation, the states of theantenna (^(a)S_(2P)) are populated and have internally converted intothe lowest excited singlet state ^(a)S₁, the excess energy istransferred to the core.

The Förster energy transfer mechanism assumes that the fluorescence(^(a)S₁→^(a)S₀) of the donor (2P antenna) overlaps with an absorptionband ^(c)S_(n)←^(c)S₀ (n=1, 2 . . . ) of the acceptor (core). Therefore,the core must possess linear absorption band(s) somewhere in the regionextending to the red from 400 nm. Exact positions of these bands aredefined by the Stokes shift of the fluorescence of the antenna relativeto its absorption at 400 nm. The FRET from the antenna to the coreresults either in the population of its singlet excited state ^(c)S₁,which is depopulated via the intersystem crossing (“isc”) to yield thetriplet state ^(c)T₁, resulting in either oxygen quenching orphosphorescence. Thus, FRET efficiency is the quantum yield of theenergy transfer transition, i.e., the fraction of energy transfer eventoccurring per donor excitation event. There are several ways ofmeasuring the FRET efficiency by monitoring changes in the fluorescenceemitted by the donor or the acceptor or as the variation in acceptoremission intensity (Clegg, R. (2009). In Gadella, Theodorus, FRET andFLIM Techniques. Laboratory Techniques in Biochemistry and MolecularBiology, Vol. 33, Elsevier. pp. 1-57). When the donor and acceptor arein proximity (1-10 nm) due to the interaction of the two molecules, theacceptor emission increases because of the intermolecular FRET from thedonor to the acceptor.

E) Two-Photon Absorption of Meso-Unsubstituted Phthalimidoporphyrins.

DAMP and TAPIP have uniquely strong two-photon absorption in the regionof 800-1000 nm. PtTAPIP has two-photon absorption (2PA) cross-sectionnear 950 nm of ˜500 Goppert-Mayer (GM) units. FIG. 6 depicts the 2PAspectra of PtTAPIP and PdTAPIP. Metal complexes of TAPIP, e.g. PtTAPIPand PdTAPIP, belong to the D_(4h) symmetry class. Therefore, theirground and excited states have definite parity: either gerade orungerade states. In D_(4h) and other systems with inversion of symmetry,the selection rules governing electric-dipole transitions by one-photon(1P) or two-photon (2P) mechanisms are mutually exclusive. For 1Pabsorption, transitions from the ground state (g-state) are allowed onlyinto u-states. For 2P absorption, transitions from the ground state areallowed only to g-states. Typically, in porphyrins 2P-allowed states(g-states) lie at high energies, such that excitation at half thatenergy (which is required for 2P absorption) overlaps with low-lyingvibronic bands of 1P allowed states. As a result, 2P absorption isovershadowed by 1P absorption into the 1P vibronic states. Furthermore,in Pt and Pd porphyrins, due to the very strong spin-orbit coupling,direct spin-forbidden absorption into the triplet state (S0→T1) gainsnon-negligible dipole strength. Consequently, 2P excitation in regularPt and Pd porphyrins becomes impossible, as energies below the tripletstate level are insufficient to promote 2P absorption into higher-lyingg-states.

In DAPIP and TAPIP, g-states are stabilized below the B-state level,where B-state is the second strongly allowed excited state, also knownas Soret state. For example, Pt and Pd TAPIP have strongly 2P-allowedg-symmetry states near 450 nm (or ˜900 nm for 2P excitation, as shown inFIG. 6). As a result, very efficient excitation into these statesbecomes possible, since there is no 1P absorption into the triplet stateor the singlet Q-state that interferes with 2P excitation at ˜900 nm.Because of their unique positioning of 2P-allowed g-states, combinedwith strong ability to form triplet states, TAPIP and DAPIP presentinterest for two-photon phosphorescence lifetime microscopy (2PLM).

F) Dendritic Phosphorescent Probes are Suitable for Oxygen Measurementsin Liquid Organic Media, e.g., Organic Solvents or Oils.

The present invention provides oxygen sensors that can be designed to bedissolved in water media (for use in biological systems) or designed tobe dissolved in organic media (for use in specialized oxygen sensors forany process in which organic media can be used, including thoserequiring organic media. This is a specific section for those that wouldbest be dissolved in organic media, for which the external shell wouldneed to different from those used in water based media. To achievesolubility of the dendritic probes in organic media the termini of thedendrimers (e.g., carboxyl groups) are amidated using 1-aminoalkanes, oresterified using 1-hydroxyalkanes to create a supramolecular hydrophobicouter layer (FIG. 8, wherein M is selected from H₂, Zn, Ni, CU, Pd, Pt,Ru, Au, Os, Ir, La. Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb,and Lu). The chemistry for this modification and the purificationprotocols closely resembles the above-discussed procedures for coveringdendrimer surfaces with PEG groups. But in accordance with addition ofthe supermolecular hydrophobic outer layer, the resulting probes showedextremely high solubility in saturated hydrocarbons, aromatic mineraloils and other organic solvents, whereas without attachment of the newoutermost layer the probe remains completely insoluble in such liquids.

In order to keep the Stern-Volmer oxygen quenching constant of the probesuitable for oxygen measurements in the physiological pO₂ range,polyarylglycine or other polyamide dendrimers (namely, polyglutamic,polyesteramide, polyarylamide) have been tested. These dendrimers foldwell in aqueous, as well as in organic environments, serving toattenuate oxygen access to the phosphorescent cores. Thus, essentiallythe same dendrimers can be used for both aqueous and hydrophobicenvironments, and only the outer layers (“supramolecular”) needs to bechanged.

In sum, methods are provided for substantially improving the currentlyavailable oxygen sensitive phosphors by increasing the quantumefficiency (phosphorescence intensity) and extending their range ofapplicability through making a class of oxygen sensors for measurementsin organic media. Thus, methods are provided in the present inventionfor synthesizing new porphyrin constructs in which the porphyrin is madeless flexible and more planar, changing with decrease internal quenchingand thereby increasing the phosphorescence emission used for oxygensensing. Additional methods are provided for structurally modifying thedendrimer used to encapsulate the porphyrin phosphor to provide forinternal quenching of any singlet oxygen formed while measuring oxygen.Moreover, in the present invention it is the outer layer that controlsthe solubility of the probe as a whole. If the probe needs to be solublein water—a water-soluble outer layer is added; whereas if the probeneeds to be soluble in organic media—an outer layer is added, making itcompatible with organics. The inventors' experiments have shown that Pdand Pt porphyrin-based Gen 2 arylglycine dendrimers (AG2), modified atthe periphery with 1-hexadecylamine possess high (ca millimolar)solubility in a variety of organic solvents (e.g., oils, saturatedhydrocarbons, aromatic mineral oils, benzene, toluene, hexane, octane,tetrahydrofurane, saturated, alkanes, mineral oil, cooking oils, oilsused in food or cosmetics, and organic liquids used in tissue andbiological analyses, and any equivalent thereof), permittingphosphorescence measurements throughout the entire ambient oxygen range:pO₂=0-160 mm Hg.

The disclosure of each patent, patent application and publication citedor described in this document is hereby incorporated herein byreference, in its entirety.

While the foregoing specification has been described with regard tocertain preferred embodiments, and many details have been set forth forthe purpose of illustration, it will be apparent to those skilled in theart without departing from the spirit and scope of the invention, thatthe invention may be subject to various modifications and additionalembodiments, and that certain of the details described herein can bevaried considerably without departing from the basic principles of theinvention. Such modifications and additional embodiments are alsointended to fall within the scope of the appended claims.

What we claim is:
 1. A phosphorescent metalloporphyrin probe, alsoreferred to as a dendritic oxygen probe, effective for oxygenmeasurement in human or animal tissue, said dendritic oxygen probecomprising a meso-unsubstituted, metallo-tetrabenzoporphyrin core,wherein the metal M is Pt, and the metallo-tetrabenzoporphyrin core hasthe following structure:

a dendrimer comprising dendrons, wherein the dendrons are peripherallyattached to the metallo-tetrabenzoporphyrin core at the 2,6 positionsindicated by “R”; and a solubilizing layer attached to the periphery ofthe dendrons.
 2. The phosphorescent probe of claim 1, wherein thesolubilizing layer is a polyethyleneglycol (PEG) layer.
 3. Thephosphorescent probe of claim 1, wherein the solubilizing layer is ahydrocarbon layer.
 4. A method of making the probe of claim 1,comprising: attaching flexible linkers terminated by functional groupsto functional groups positioned directly on a meso-unsubstituted,metallo-tetrabenzoporphyrin core, wherein the metal is Pt, and the Ptmetallo-tetrabenzoporphyrin core has the following structure:

wherein “R” indicates the attachment of dendrons to the core at 2, 6positions; attaching dendrons to the functional groups of the linkers;attaching solubilizing residues to peripheral functional groups on thedendrons to form a solubilizing layer.
 5. A method of using themetalloporphyrin probe of claim 1, the method comprising the steps of:dissolving the probe in the blood, and exciting the probe usingappropriate illumination, wherein the probe exhibits strong lightabsorption in a near infrared region of a spectrum and upon excitation,provides quantum yields of phosphorescence 5-10 fold higher, as comparedwith a corresponding tetraarylporphyrin when measured at the sametemperature and conditions.
 6. A method of improving dendrimerization ofa meso-unsubstituted, metallo-tetrabenzoporphyrin core, wherein themetal M is Pt, and the metallo-tetrabenzoporphyrin core has thefollowing structure

wherein “R” indicates the attachment of dendrons to the core at 2, 6positions, the method comprising the steps of: attaching flexibleextension linkers terminated by functional groups to functional groupspositioned directly on the metallo-tetrabenzoporphyrin core, and therebyspacing reaction sites at a sufficient distance apart to cause enhancingof nucleophilic attack and reduce contamination by imperfect dendrimersin a yield and purity enhancing step.
 7. A method of using the improveddendrimerization method of claim 6 using a metalloporphyrin probe,wherein the attachment of the flexible extension linkers provides 1-2times higher-yield of synthesis of pure monodisperse dendritic oxygenprobes as compared to corresponding phosphorescent porphyrinssynthesized by the same steps of hydrolysis of peripheral ester groups,attachment of dendrons and hydrolysis of their peripheral esters andPEGylation, but without attaching flexible extension linkers to anchorgroups during synthesis at the same temperature and conditions.
 8. Aphosphorescent metalloporphyrin probe having improved dendrimerization,also referred to as a dendritic oxygen probe, effective for oxygenmeasurement in human or animal tissue, said dendritic oxygen probeproduced by the method of claim 6, the probe comprising added flexibleextension linkers attached to anchor groups on either meso-aryl rings ordirectly on a metalloporphyrin macrocycle, thereby spacing reactionsites at a sufficient distance apart to enhance nucleophilic attack andreduce contamination by imperfectly reacted dendrimers after a reactiontime of only the overnight period at room temperature.
 9. A method ofcausing a dendritic phosphorescent probe suitable for oxygenmeasurements in aqueous liquids, the method comprising: adding a threedimensional supramolecular hydrophilic outer layer to the termini of adendrimer having a meso-unsubstituted, metallo-tetrabenzoporphyrin core,wherein the metal M is Pt, and the metallo-tetrabenzoporphyrin core hasthe following structure:

wherein “R” indicates the attachment of dendrons to the core at 2, 6positions.
 10. A method of causing a dendritic phosphorescent probesuitable for oxygen measurements in organic liquids, the methodcomprising: adding a three dimensional supramolecular hydrophobic outerlayer to the termini of hydrophobic dendrimers having ameso-unsubstituted, metallo-tetrabenzoporphyrin core, wherein the metalM is Pt, and the metallo-tetrabenzoporphyrin core has the followingstructure:

wherein “R” indicates the attachment of dendrons to the core at 2, 6positions; causing the molecule to remain folded, but to become highlysoluble in organic solvents selected from the group comprising oils,saturated hydrocarbons, mineral oil and aromatic mineral oils, benzene,saturated alkanes, tetrahydrofuran (THF), toluene, hexane, octane,cooking oils, oils used in food or cosmetics, and organic liquids usedin tissue and biological analyses; and enabling oxygen measurementstherein.
 11. A dendritic phosphorescent probe produced by the method ofclaim 10, comprising a selectably added three dimensional supramolecularhydrophobic outer layer attached to the termini of the hydrophobicdendrimers, thereby causing the molecule to remain folded, and to behighly soluble in organic solvents selected from the group comprisingoils, saturated hydrocarbons, mineral oil and aromatic mineral oils,benzene, saturated alkanes, tetrahydrofuran (THF), toluene, hexane,octane, cooking oils, oils used in food or cosmetics, and organicliquids used in tissue and biological analyses.
 12. A method forsynthesizing a meso-unsubstituted tetraphthalimidoporphyrin, the methodcomprising: reacting a sulfolene bearing an electro-withdrawing groupwith isocyanoacetate to yield a sulfolenolpyrrole ester, synthesizing amaleimide from maleic anhydride and amine R—NH₂, where group R is analkyl or an aryl, performing a Diels-Alder reaction of thesulfolenolpyrrole ester and the maleimide to generate acyclohexenopyrrole having ester groups, cleaving ester groups from thecyclohexenopyrrole to yield a pyrrole, reacting the pyrrole withformaldehyde in the presence of an acidic catalyst to yield a product,oxidating the product of the reaction of the pyrrole with formaldehydeto generate a tetracyclohexenoporphyrin, metallating thetetracyclohexenoporphyrin with a metallic salt having metal M, whereinmetal M is selected from the group comprising Zn, Ni, Cu, Pd, Pt, Ru,Au, Os, Ir, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, andLu, to produce a metalloporphyrin, and oxidizing the metalloporphyrin.13. The method of claim 12, wherein the group R of amine R—NH2 is analkyl or an aryl selected from the group comprising selected from thegroup comprising methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl,octyl, decyl, dodecyl, phenyl, tolyl, xylyl, hydroxyphenyl,dihydroxyphenyl, aminophenyl, sulfophenyl, and bromophenyl.
 14. Themethod of claim 12, wherein the group R of amine R—NH2 is a2,6-disubstituted aryl groups.
 15. The method of claim 12, wherein theacidic catalyst is trifluoroacetic acid.
 16. The method of claim 12,wherein the metalloporphyrin is oxidized withdichlorodicyanobenzoquinone (DDQ) or its analogues.
 17. A method forsynthesizing a meso-unsubstituted tetraphthalimidoporphyrin, the methodcomprising: reacting a sulfolene bearing an electro-withdrawing groupwith isocyanoacetate to yield a sulfolenolpyrrole ester, synthesizing amaleimide from maleic anhydride and amine R—NH₂, where group R is analkyl or an aryl, performing a Diels-Alder reaction of thesulfolenolpyrrole ester and the maleimide to generate acyclohexenopyrrole having ester groups, cleaving ester groups from thecyclohexenopyrrole to yield a pyrrole, reacting the pyrrole withformaldehyde in the presence of an acidic catalyst to yield a product,oxidating the product of the reaction of the pyrrole with formaldehydeto generate a free-base tetracyclohexenoporphyrin, oxidizing thetetracyclohexenoporphyrin to yield a free-base TAPIP, metallating thefree-base TAPIP with a metallic salt having metal M, wherein metal M isselected from the group comprising Zn, Ni, Cu, Pd, Pt, Ru, Au, Os, Ir,La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu, toproduce a metalloporphyrin,
 18. The method of claim 17, wherein thegroup R of amine R—NH2 is an alkyl or an aryl selected from the groupcomprising methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl,decyl, dodecyl, phenyl, tolyl, xylyl, hydroxyphenyl, dihidroxyphenyl,aminophenyl, sulfophenyl, and bromophenyl.
 19. The method of claim 17,wherein the group R of amine R—NH2 is a 2,6-disubstituted aryl groups.20. A method for synthesizing a meso-unsubstituteddiphthalimidoporphyrin, the method comprising: reacting a sulfolenebearing an electro-withdrawing group with isocyanoacetate to yield asulfolenolpyrrole ester, synthesizing a maleimide from maleic anhydrideand amine R—NH₂, where group R is an alkyl or an aryl, performing aDiels-Alder reaction of the sulfolenolpyrrole and the maleimide togenerate a cyclohexenopyrrole having ester groups, introducing thecyclohexenopyrrole having ester groups into reaction with itself toyield a dipyrromethane-diester, de-esterifying, decarboxylating, andcondensing the dipyrromethane-diester with diformyldipyrromethane havingsubstituents R₁ and R₂ to generate a dicyclohexenoporphyrin, metallatingthe dicyclohexenoporphyrin with a metallic salt having metal M, whereinmetal M is selected from the group comprising Zn, Ni, Cu, Pd, Pt, Ru,Au, Os, Ir, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, andLu, to produce a metalloporphyrin, oxidizing the metalloporphyrin. 21.The method of claim 20, wherein the group R of amine R—NH2 is selectedfrom the group comprising methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl, octyl, decyl, dodecyl, phenyl, tolyl, xylyl, hydroxyphenyl,dihydroxyphenyl, aminophenyl, sulfophenyl, and bromophenyl
 22. Themethod of claim 20, wherein the group R of amine R—NH2 is a2,6-disubstituted aryl group.
 23. The method of claim 20, wherein themetalloporphyrin is oxidized with dichlorodicyanobenzoquinone (DDQ) orits analogues.
 24. A method for synthesizing a meso-unsubstitutedtetraphthalimidoporphyrin, the method comprising: reacting a sulfolenebearing an electro-withdrawing group with isocyanoacetate to yield asulfolenolpyrrole ester, synthesizing a maleimide from maleic anhydrideand amine R—NH₂, where group R is an alkyl or an aryl, performing aDiels-Alder reaction of the sulfolenolpyrrole and the maleimide togenerate a cyclohexenopyrrole having ester groups, introducing thecyclohexenopyrrole having ester groups into reaction with itself toyield dipyrromethane-diester, de-esterifying, decarboxylating, andcondensing the dipyrromethane-diester with diformyldipyrromethane havingsubstituents R₁ and R₂ to generate a free-base dicyclohexenoporphyrin,oxidizing the free-base dicyclohexenoporphyrin to yield a free-baseDAPIP, metallating the free-base DAPIP with a metallic salt having metalM, wherein metal M is selected from the group comprising Zn, Ni, Cu, Pd,Pt, Ru, Au, Os, Ir, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm,Yb, and Lu, to produce a metalloporphyrin.
 25. The method of claim 24,wherein the group R of amine R—NH2 is selected from the group comprisingmethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, decyl,phenyl, tolyl, xylyl, hydroxyphenyl, dyhydroxyphenyl, aminophenyl,sulfophenyl, and bromophenyl.
 26. The method of claim 24, wherein thegroup R of amine R—NH2 is a 2,6-disubstituted aryl groups.